In order to prevent risks of complications, e.g. during a transfusion, in particular blood group incompatibilities, viral and/or bacterial contaminations it is known to perform a variety of laboratory tests on the donor and patient blood in order to make available components which are compatible in a blood group serological sense with the recipient and are free of known transmittable pathogens. The respective serological tests as a rule include the determination of the blood group of the donor and the recipient, in particular of blood groups of the blood group systems ABO, Rh and Kell, serum cross-checks on the donor and recipient, antibody detection tests in respect of irregular antibodies of the donor and recipient as well as antibody identifications in the case of the recipient where irregular antibodies are present. The detection of antibodies against thrombocytes and or lymphocytes is performed likewise in the context of transfusions and transplantations.
Infection serological tests on the donor include in a known manner the routine determination of antibodies, in particular against HIV-I, HIV-H, against HCV against treponema pallidum (syphilis), as well as the determination of the hepatitis B surface antigen (=HbsAg: hepatitis surface antigen).
In blood group serological diagnostics, parameters are generally tested which are of particular relevance in the context of transfusions or of morbus haemolyticus neonatorum (Mhn). This includes inter alia the detection of antigens on the surface of erythrocytes which are characteristic for the blood groups (blood group determination). Further important antigen systems are present also on thrombocytes, granulocytes, lymphocytes which likewise play a role in the context of transfusions and transplantations. In the event of thrombocytes and granulocytes antigen non-identity between mother and fetus pathological symptoms may occur with neonatants comparable with Mhn. In addition the detection of regular blood group antibodies (isoagglutinine) and of irregular blood group antibodies in serum or plasma is applicable here.
The isoagglutinines or regular antibodies are acquired particularly by humans soon after their birth and correspond to the respective blood group of the ABO system. They are directed against those blood group antigens A or B which the individual itself lacks, i.e. persons which have the blood group A have anti-B, persons of blood group B have anti-A persons of blood groups O have anti-A and anti-B; persons of blood group AB have no isoagglutinine. The regular antibodies are also known as “complete” because they are able to directly agglutinate erythrocytes in NaCl medium.
The irregular or allo-antibodies, in contrast to the isoagglutinines are acquired by immunizations in later life, in particular by transfusion or pregnancy. For that reason most humans have no irregular blood group antibodies. The transfusion relevant irregular antibodies are as a rule heat reactive and belong mostly to the IgG class. In contrast to the regular antibodies they are not able to directly agglutinize erythrocytes in a NaCl medium.
It is known that for determining blood groups the erythrocytes of the persons to be tested, (donors or recipients) are brought together with reagents which contain blood group specific antibodies. Generally these tests are performed in the liquid state, in which by mixing of an erythrocyte containing sample with a sample containing antibodies directed against a specific blood group characteristic a testing batch is produced. The testing batch is then incubated over a defined period and under defined conditions and after conclusion of the incubation, either directly or after a centrifugation step, is tested visually or by optical methods for a possible agglutination or adsorption of the erythrocytes. The predominant end point measurement in blood group serology is still the hemaglutination test. For each blood group to be determined a separate batch must be pipetted, i.e. e.g. for the determination of the nine most important blood groups A, B, D, C, c, E, e, Cw and K, nine separate batches are needed, without counting any control.
For the serum cross-check cell reagents with known ABO blood groups (A1, A2, B, 0) are used in a known manner which are incubated with the serum or plasma of the person to be tested. After a centrifugations step a possible agglutination of the erythrocytes is tested for visually or by optical methods. For a serum cross-check with the aforesaid test cells it is conventionally necessary to pipette four batches.
In order to search for irregular antibodies panels are generally used comprising two or three blood group O cells the combined antigen profile of which contains the most important antigens, in particular the blood group systems Rh, Kell, Duffy, Kidd, MNS, P, Lewis, Lutheran. The cell reagent is brought together with the serum or plasma of the person to be tested, is incubated and after a step of centrifugation is tested visually or by optical methods for any agglutination of the erythrocytes. Two to three batches must be pipetted for testing one patient sample.
In order to identify the irregular antibodies which as a rule takes place after a positive antibody detection test, panels comprising up to 16 blood group O cells are used the antigen profile of which covers the most important antigens, in particular the blood group systems Rh, Kell, Duffy, Kidd, MNS, P, Lewis, Lutheran in an exactly predetermined manner. The cell reagent is brought together with the serum or blood plasma of the person to be tested and by visual or optical methods is tested for any agglutination of the erythrocytes. For testing a patient sample up to 16 batches must be pipetted.
Because most transfusion relevant irregular antibodies are of the IgG type and are therefore incomplete the reactions for antibody detection and identification as described must as a rule be reinforced in order to be able to detect the end point of the hemaglutination. The most common reagent for this purpose is a polyclonal anti-humanglobulin reagent to which frequently anti-complement antibodies have been added (typically anti-C3d and/or anti-C3b).
A commonly used method for detecting thrombocyte antibodies is the so-called MAIPA test (monoclonal antibody immobilization of platelet antigens). In this case test thrombocytes are incubated with the serum to be tested. After a rinsing step, incubation is performed with a monoclonal, e.g. mouse antibody which is specific for a particular thrombocyte glycoprotein. The thrombocytes are thereafter subjected to lysing and the diluted lysate is introduced into a reaction vessel coated e.g. with goat anti-mouse antibodies of a microtitration panel. The goat anti-mouse antibody binds the mouse antibody and the thrombocyte glycoprotein human antibody complex attached thereto. The human antibody is tested for by the addition of an enzyme-conjugated goat anti-human IgG.
With conventional diagnostic tests it is possible only to either determine cellular or plasmatic parameters. In order to determine blood components it is invariably necessary to first separate cells from plasma.
Lateral flow tests nowadays are frequently applied as quick tests e.g. as pregnancy tests, for determining infection markers or for drug screening. A lateral flow test device in a known manner includes a rigid support on which an application zone for the sample to be tested is provided, a separating membrane, on which bonding elements, e.g. catcher antibodies or antigens are bound and on which the bonding reactions can be detected, and a suction generating absorption region which causes the sample to be tested to flow in a linear manner through the separating membrane.
Test membranes of conventional lateral flow tests are generally described involving a chromatography-like separation. The analyte in the sample binds specifically to the bonding elements fixed in a membrane which as a rule are present in consecutive or superimposed bands serving as indicator zones. The binding complex is rendered visible by indicator particles which as a rule are already present in the device in dehydrated form in a conjugate liberation pad. The conjugate liberation pad is provided between the application zone and the membrane. The pre-coated colored indicator particles are coated for example with an antibody directed against the analyte to be tested for.
The conventional lateral flow test format corresponds to a so-call “sandwich assay”, in which both the indicator zone as well as the indicator particles are coated with a ligand aimed at the analyte tested for, normally an antibody. In that context the ligand (bonding element) is immobilized on the membrane. The detector reagent, normally an antibody bonded to a colored polystyrene particle or to colloidal metals, is deposited in the conjugate liberation pad in a leachable manner. This bonding complex serves as indicator particle. Once the sample to be tested has been applied it very rapidly wets the conjugate liberation pad, whereby the indicator particles are mobilized. The indicator particles migrate with the liquid front along the porous membrane. An analyte present in the sample becomes bonded by the antibody coupled to the indicator particle. As the sample passes the indicator zone, the analyte/indicator particle complex in the indicator zone is immobilized by reaction of the analyte with the antibody bonded in the indicator zone, resulting in a visible signal.
A further known test format for small analytes comprising but a single antigenic determinant, incapable of simultaneously bonding two antibodies, is the so-called “competition assay”. The detector reagent bonded to the indicator particle is normally a molecule identical to or analogous with the analyte. The indicator particles are deposited in the conjugate liberation pad. The indicator particles migrate with the liquid front along the porous membrane. If the sample contains the analytes, and if the indicator particles (which effectively likewise contain analyte) pass the indicator zone, part of the analyte molecules in the sample bond to part of the indicator particles. The more analyte is present in the sample the more effective will it compete with the bonding of the indicator particle and the weaker will the signal become.
According to the prior art these indicator particles are predominantly composed of colloidal gold or of polystyrene, manufactured and coated according to methods known to the skilled person. In the typical lateral flow tests formats the analytes are determined indirectly. In this context a direct determination of an analyte denotes that the analyte is already bonded naturally to the indicator particle (e.g. erythrocyte). In the more common situation of indirect determination of the analytes the sample to be tested as a rule contains a non-cellularly bonded, e.g. plasmatic component as the analyte and, besides the sample to be tested, two reagent components are required, i.e. indicator particles and a bonding element. In the indirect determination the analyte initially bonds to the indicator particle dissolved out of the conjugate liberation pad, before this complex becomes immobilized in the indicator zones with the bonding element by way of a second reaction.
When using conventional lateral flow tests with erythrocytes as indicator particles which have been bonded to the analytes to be determined, for example blood group specific antigens, it is at present usual for antibodies to be provided in the indicator zones against corresponding blood group antigens serving as bonding elements in successive or superimposed bands in but a single flow track such as for example anti-A, anti-B against the Rh blood group system. In this context conventional lateral flow tests suffer from the disadvantage that the erythrocytes bonded to the antibodies form a flow barrier against the analytes still to be tested for, for example further cell associated antigens, in a sample. Due to agglutination or adsorption of cells in a band of bonding elements arranged proximally to the application zone, additional analytes, in particular cells or cell fragments in the sample to be tested, can no longer be separated unimpededly and visibly and can therefore not be tested for unambiguously or completely. For example in a person who is blood group AB Rh D positive this may result in a weakening or elimination of the B and the D bands, which may result in a faulty interpretation of being blood group A Rh negative. For that reason it was hitherto not possible, specifically in blood group serological diagnostics to employ a lateral flow test with more than one indicator zone. In order to determine a plurality of, in particular cellular and plasmatic blood group parameters, it is to date necessary to conduct single parameter tests separately.